High throughput X-Ray powder screening of materials for useful properties reduce development times by up to orders of magnitudes, at a fraction of costs. For example, ability to unequivocally differentiate one crystallographic phase from another has proven to be the greatest asset in the pharmaceutical field: powder X-Ray diffraction patterns of each crystalline form contain characteristic sets of peaks unique to that form, allowing the patterns to be used as fingerprints. Fully automatic powder crystal data evaluation may allow speedy (pattern analyzed in minutes) and cost effective handling of big amount of information obtained. Such high throughput techniques have strong impact on quality control of industrial products.
In many modern scientific and industrial applications, it is necessary to be able to study samples at the nanometer scale, where most materials are ordered, often crystalline. Their physical properties depend on the crystal structure. Unfortunately, the traditional method for atomic structure determination, X-ray crystallography, cannot be used for single crystals with sizes in the nanometer range, either because the limit size for single crystal structure determination with modern Synchrotron sources is above 5 microns, or because powder X-Ray patterns show extremely poor resolution (e.g. peak broadening and overlapping) at decreasing crystallite size (nm). This simple fact limits the contribution of X-ray crystallography and consequently to any attempt of high throughput analysis to nanoscience, a scientific area crucial in many fields, from semiconductors to pharmaceuticals and proteins. Therefore, compounds that only exist at nanocrystalline state are usually out of reach for the X-ray diffraction methods of structure determination and as a result, such nanostructures, despite their given importance, are unknown. The resulting lack of knowledge on the underlying structure-property relationships often prohibits breakthrough developments in a whole research sector or may cause fatal delays in the cycle of further product development.
The resulting lack of knowledge of the underlying structure-property relationships often prohibits breakthrough developments in a whole research sector, or may cause fatal delays in the cycle of further product development.
Electron diffraction (ED) is the method of choice to solve the structural problems at nanocrystal size, as electrons interact about 103 to 104 times stronger with matter than X-rays and, therefore, are ideal for ED of crystals in the nanometer sized range. Nanocrystalline samples can be studied both by electron microscopy and electron diffraction. Transmission electron microscopy (TEM) which uses an electron beam in transmission mode, gives a direct image of the structure, but suffers from lens distortions and limited resolution. This is contrary to the use of an electron beam in electron diffraction techniques which can go to very high resolution e.g. 0.5 Ångström or even higher, but suffer from dynamic scattering. However, it is currently not possible to use ED intensities directly (unlike X-Ray techniques) to solve crystal structures; these drawbacks currently limit general use of ED as reliable technique for nanocrystal structure analysis.
Solving the three dimensional structure of any nanocrystal requires collection of 3 dimensional (3D) intensity data from as many zone axes (ZA) as possible from the same crystal. For many, less symmetrical structures e.g. monoclinic, triclinic crystals, it is important to tilt the crystal to obtain such ZA through a wide range of angles (for example from −40° to 40°). Conventional TEM used in diffraction mode do not always allow high-tilt angles due to restrictions caused by the geometry of the specimen holder and polepiece gap of the objective lens. This causes severe limitations in the attainable resolution of the 3D data, (missing cone problem). As a consequence, 3D data acquisition for any nanocrystal is an extremely time-consuming task—it may take up to days even for experienced researchers to obtain a good quality data set—which makes the method highly unattractive and inappropriate for routine investigations. The situation becomes even worse when studied nanocrystals are electron beam sensitive organics; in that case, even when low dose conditions such as cryo-cooling are applied to minimise beam damage, beam sensitivity makes it impossible to conserve crystal structural integrity for more than few minutes/seconds, making therefore the whole time-consuming standard procedure of single crystal orientation and data acquisition (that normally lasts tens of minutes) difficult or impossible.
Thus, traditional TEM ED data collection techniques are generally limited; the standard procedure is very time consuming, it does not allow collection of a complete 3D-ED intensities on the same single crystal and the diffraction intensities which carry information about the crystal structure are of poor quality, due to multiple/dynamical scattering of the electrons.
In this context, the electron diffraction precession method (EDPM) developed by Roger Vincent & Paul Midgley (Vincent R., Midgley, P. Double conical beam-rocking system for measurement of integrated electron diffraction intensities. Ultramicroscopy 53:271-282, 1994) plays a vital role, since it made it possible for the first time significantly to reduce dynamic diffraction effects. In this technique the electron beam is tilted by a small angle, typically 1-3 degrees, and then rotated around the TEM optical axis. In this way a volume of reciprocal space is recorded (integration over excitation error), and more importantly, the multiple/dynamic scattering is greatly reduced, since only a small number of reflections are excited at any time.
An alternative technique for collecting complete 3D electron diffraction data from single nanocrystal, known as electron diffraction rotation method (EDRM), has been proposed (PCT/SE2007/050853) which also reduces the multiple/dynamic scattering. Electron rotation can be achieved by a device rather similar to the one that achieves electron precession. The main difference is that the electron beam does not rotate a circle, but rather follows a straight line, like a pendulum. This line can be along the x-direction, along the y-direction or along any diagonal in between. In order to handle partially recorded reflections, the data must be collected in a number of small angular steps. For example, each scan may have a rotation of only +/−0.5 degrees along a line. The next scan will follow on from exactly where the previous stopped, i.e. from +0.5 to +1.5 degrees, with the next one +1.5 to +2.5 degrees and so on. One such series of rotation patterns can total up to about 6 degrees; the exact range is limited by the design of the specific model of electron microscope, then the crystal will be tilted by a few degrees and the data acquisition will start again. This way, a complete 3D diffraction set may be obtained in diffraction tomography-like mode. Such device for EDRM can rotate the electron beam by a predetermined but variable angular rotation range, and along any direction.
However, one of the drawback of the EDRM technique and the aforementioned EDPM technique is that tilting simultaneously through the whole angular range for 3D data collection and aligning the TEM holder is a very time consuming procedure In addition, due to electron beam sensitivity for crystals useful in pharmaceutical industry and protein nanocrystals, even under low dose conditions and LN2 cooling, only a limited number of ED patterns (e.g. 1-3) can be obtained from each crystal. Moreover, both EDPM and EDRP techniques rely on 3D diffraction data collection by manual or automatic (through computerized TEM holders) tilts of a particular single nanocrystal around a selected crystallographic axis.
An automated electron diffraction tomography procedure (ADT) has been developed which combines nanobeam ED and STEM imaging using a high angular annular dark field detector (HAADF); the use of the latter detector permits a significant reduction in beam damage in comparison with more conventional approaches to diffraction. With ADT, a typical approach during experimental work is to find a suitable crystal, select it with a marker (spot or allowed area) and tilt it, according to a given sequence, around the goniometer axis; there is no need for crystallographic axis to be oriented along the goniometer axis. Automated data processing routines provide automated cell parameter determination after a peak search. Resulting (partial) 3D diffraction data network allows the detection of crystal cell, effects such as partial disorder, twinning, polymorphs and superstructures; measuring ED intensities, even from non-complete ED network may allow to solve individual crystal nanostructures. However, ADT is equally time consuming technique as previous EDPM and EDRM techniques.
It is clear from the prior art that techniques of the art for nanocrystal structure analysis based on electron diffraction are time consuming and use long exposure protocols to obtain full range of tilt angles. A more time-efficient procedure is necessary, which lends itself well to high throughput measurement.